Grass treatment

ABSTRACT

A mycorrhiza have growth-retardant activity for  Poa annua  and may be used to control the growth of  P. annua  in high quality sport or amenity turf consisting mainly of Agrostis or Festuca species. Strains of VA mycorrhiza selected from genera Glomus, Acaulospora, Entrophosphora, Gigaspora, Scutellospora and Scierocytis may be used. The VA mycorrhiza is applied to the soil as a formulated product in which the VA mycorrhizae constitute the principal component having biological activity on the growth of turf grass. Dose rates of inoculum having an MPN of from about 200 To 1000 or more propagules per gram are effective and may be formulated with a clay or other solid carrier. The amount of formulated product applied to the turf may be from about 5 g/m 2  to about 1 kg/m 2 . The infective prop are selected from fungal spores, mycelium, hyphae, and fragments of mycorrhiza infected roots. These may be applied to sport or amenity turf, or other grassy area in need of  P. annua  control, or an area in which high quality turf-grass is to be laid or sown.

This invention relates to methods and compositions for improving thequality of turf grass. More particularly, the invention is concernedwith the control, reduction, or elimination of undesirable grasscomponents of turf, especially of high quality turf consisting mainly ofBentgrass such as Agrostis stolonifera or Festuca species.

Poa annua or Annual Meadow grass (AMG) (also called Annual Bluegrass) isthe most troublesome weed of bent grass putting greens in countries asdiverse as the United Kingdom, the United States of America, andAustralia. P. annua is generally considered to be undesirable in puttinggreens because it is susceptible to abiotic stress, particularly wateravailability, as well as succumbing to a number of fungal diseases. Inaddition, if a green is composed of patches of AMG and bent grass, thesurface is not as uniform as many players would wish it to be.

Some turf managers attempt to control P. annua. The most widely usedcontrol method is herbicide application, and a number of compounds havebeen tested against the species with varying success. However,application of pesticides can be costly, and they may present a problemof groundwater pollution if the green is well irrigated. In addition,there may be associated health risks to golfers or greens staff.Therefore, if AMG is to be controlled on putting greens, a more natural,environmental approach is called for.

The present inventor has previously reported that the abundance of AMGin a golf green may be negatively related to the abundance of fungi inthe soil (Gange A. C. 1994, Subterranean insects and fungi: hidden costsand benefits to the greenkeeper. In Science & Golf II, Proceedings ofthe World Scientific Congress of Golf, eds. A. J.Cochran and M. R.Farrally, 461-466, London E. and F. N. Spoon ). The fungi concerned werevesicular-arbuscular mycorrhizas which are generally abundant in naturalplant communities. It was found that the fungi were very low inabundance in golf turf, but when they were common in the soil, there wasless AMG in the sward, and vice versa. The original explanation for thisrelation was that as bentgrass is considerably more strongly mycorrhizalthan AMG, then in greens where fungal abundance is high, the bentgrassis more vigorous, and therefore is able to out-compete the AMG.

It has now been found that VA mycorrhizae have a directgrowth-controlling effect on Poa annua and therefore provide aneffective means of control of P. annua in turf grass consisting of highproportions of Bentgrass, or Festuca species. This is an unexpectedfinding for two reasons. First, P. annua is usually stated in theliterature to be non-mycorrhizal or at most weakly mycorrhizal.Secondly, whilst it has now been found that VA mycorrhizae do colonisePoa annua roots, the colonisation retards rather than encourages growth.Moreover, this effect has been found to be independent of phosphoruslevels in the soil.

The present invention is therefore based on the discovery of a noveltechnical effect of VA mycorrhizae which leads to a method of treatmentof turfgrass which has not been contemplated hitherto. It is to beunderstood that the application of VA mycorrhizae, either to grasslandor soil in preparation for grass sowing, in accordance with thisinvention is for the specific intention of suppressing Poa annua, asdistinct from any other purpose e.g. to stimulate growth or for therecovery of damaged turf. Preparations for use according to theinvention do not require the presence of any other bacteria or otherorganisms or biologically active materials, although these are notexcluded if desired as incidental to the primary purpose of theinvention.

It is especially important that the composition applied to turf containsvery little of, and is preferably substantially free of, otherbiological materials which could promote growth of P.annua and thereforecompete with the VA mycorrhiza and conflict with the objectives of thepresent invention. For example, because P. annua dominates turf in areasof high phosphate application, it is highly desirable to avoid thepresence in the composition of significant amounts of bacteria whichrelease P from phosphate. The deliberate addition of suchphosphate-solubilizing bacteria to the composition is stronglycontra-indicated. Preferably, therefore, apart from minimal amounts ofother organisms which may be present due to the use of conditions ofproduction which are not totally sterile, the VA mycorrhiza fungi usedconstitute the sole or principal organismic component of thecompositions of the present invention.

The present invention comprises the use of a VA mycorrhiza as agrowth-retardant for Poa annua. The term ‘VA mycorrhiza’ is used hereinto cover all soil-borne fungi which form arbuscules in obligatemutualistic associations with terrestrial plants. Typicalrepresentatives of the vesicular-arbuscular fungi are found among thegenera Glomus, Acaulospora, Entrophosphora, Gigaspora, Scutellospora andSclerocytis. Exemplary strains are Glomus fasciculatum, Glomuscaledonium, Glomus mosseae, Glomus versiforme, Glomus intraradices, andGlomus etunicatum. One or more strains of VA mycorrhiza may be useddepending on the quality of the turfgrass to be treated. Preferredstrains are those of Glomus mosseae, caledonium, fasciculatum, andversiforme. VA mycorrhizae are widely available organisms and may beobtained, for example, from the International Culture Collection of VAMfungi (INVAM), Florida, United States of America.

To produce VA mycorrhizal inoculum for the purposes of the presentinvention all infective structures of the fungus can be used, includingspores and mycelium produced inside or outside the host root. Infectedroots and infected substrates can also be used. Inoculum can be used inthe form of slurries, gels, pellets, infected soil, or sporeformulations. Especially effective formulations are the claycarrier-based formulations containing the four preferred strainsindicated above e.g. those available commercially under the RegisteredTrade Mark ‘VAMINOC’ (The MicroBio Group Ltd, Whittlesford, Cambridge.UK), such as VAMINOC 8/16, VAMINOC 30/60, AND VAMINOC-T. Inoculantparticle sizes can be up to 2 mm or 4-8 mm. Alternative carriers can beselected from, for example, silica gel, bleaching earths, pumice,bauxite, attapulgite, vermiculite, calcined montmorillinite, soil, peat,sand or any other substantially chemically inert material with asuitable porous structure.

The grass which may be treated according to the present invention may bea sport or amenity turf which is already established and which containsunacceptable proportions of P. annua. Turf which is mown regularly isparticularly suitable for treatment. Alternatively the treatment may beapplied to ground on which turf is to be laid or sown e.g. a sand/peatbased green sown with bentgrass or a bentgrass/Festuca mixture.

Desirably, the inoculum infectivity, as measured by the “most probablenumber” (MPN) technique, corresponds to MPN values of from at least 200or 500 up to 1000 propagules or more per gram. Application of inoculumat a rate of at least 5 or 10 g/m² is effective but higher dosages maybe used if preferred, e.g. up to about 100 to 250 g/m². Even higherdoses, for example up to and in excess of 1 Kg/m², are envisaged incertain applications. The inoculum can be watered into an establishedgreen or introduced into the root zone. Application in weather which iseither very warm or very cold is inadvisable. The turfgrass may be agolf green, a bowling green, a lawn, or any other kind of grassy arearequiring treatment according to the invention. The invention is ofparticular importance in improving or maintaining the quality of turfconsisting mainly of Agrostis Spp or Festuca Spp.

The invention also comprises a method of controlling or reducing theamount of P. annua in turfgrass which comprises applying to theturfgrass a growth-inhibiting strain of VA mycorrhiza. Preferably, acomposition containing infective propagules is applied to the turfgrass,the composition containing propagules in sufficient quantities toeffectively colonise plant roots. Infective propagules can be selectedfrom, but are by no means limited to, fungal spores, mycelium, hyphae,and fragments of mycorrhizal infected roots. Experiments which haveestablished the growth retarding activity of VA mycorrhizae aredescribed below and illustrated in FIGS. 1 to 7 of the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a graphically shows a significant reduction of growth of Poa annuain response to Vaminoc inoculation.

FIG. 1b photographically shows the detrimental effect of VA-mycorrhizaon Poa.

FIG. 2 shows that the weights for Poa annua trays not inoculated withVaminoc were higher than those for the inoculated trays.

FIG. 3 shows that grass treated with the Vaminoc-T mycorrhiza inoculanthad significantly more total grass touches.

FIG. 4 shows that grass treated with Vaminoc-T had more touches ofAgrostis.

FIG. 5 shows that plots treated with Vaminoc-T had fewer touches of Poaannua than the controls.

FIGS. 6 and 7 show the relationship between Vaminoc-G application andincidence of Poa annua on golf greens.

INITIAL EXPERIMENTS

In July 1997, three soil cores, each 1.5 cm diameter were taken from all18 greens on a golf course. Each core was examined under a binocularmicroscope and the number of tillers of each grass species counted.Roots of the grass species were then washed free of soil and placed onmicroscope slides. They were then examined under an epifluorescencemicroscope fitted with a UV bulb, under which conditions, the arbuscules(the only definitive structure of the mycorrhiza) autofluoresce.Mycorrhizae were quantified with the cross-hair eye piece method ofMcGonigle et al., 1990. Differences between greens in the abundance ofAMG were tested for with Kruskall-Wallis one way Analysis of Varianceand the relation between mycorrhizal colonisation and AMG abundanceexamined with linear regression.

Laboratory Experiment

Eighty flower pots, each measuring 7 cm×7 cm were filled with a mixtureof 80% sand, 20% fen loam soil and sown with Agrostis stolonifera at therate of 40 g m⁻². Half of the pots were inoculated with 8.7 g ofVaminoc-T, a four species mixture of mycorrhizal fungi (The MicroBioGroup Ltd, Whittlesford, Cambridge, UK) equivalent to 25 g per liter ofsoil. Ten mycorrhizal and ten non-mycorrhizal pots then received 0.2 gof AMG (equivalent to 157,000 seeds m⁻²), ten of each treatment received0.4 g of AMG seed (314,000 seeds m⁻²) and ten of each treatment, 0.6 gof AMG seed (471,000 seeds m²). There were also ten pots of eachmycorrhizal treatment which received no AMG seed. Therefore, in totalthere were eight treatments (2 levels of mycorrhiza×4 levels of AMG),with ten replicates of each, giving 80 pots.

Pots were maintained in a Constant Environment room at 20 C. for threemonths, by which time a close sward had developed. The cutting height ofthe grass was gradually reduced to 8 mm and at each cut the clippingswere removed and dried to constant weight in an oven. Fertiliser wasapplied to mimic that recommended for an establishing green. At the endof the experiment the total biomass produced in each pot was calculated.The effect of adding AMG and the mycorrhiza on biomass production wasanalysed with a nested Analysis of Variance.

Results

There was an expected effect of AMG addition on total biomass, but nooverall effect of mycorrhizal addition. However, there was a highlysignificant interaction between AMG and mycorrhiza (F_(3.72)=4.5,p<0.01). This was because when no AMG was added to the pots, addition ofmycorrhiza increased the biomass of the bent grass. However, in all AMGaddition treatments, the mycorrhiza did not increase the total biomass,and in some cases, significantly reduced it. These data therefore implythat mycorrhiza had a positive effect on the bentgrass, but a negativeeffect on the AMG. This can be seen by comparing pairs of treatments inthe experiment. Thus, the addition of 0.2 g of AMG to non mycorrhizalpots caused an increase of 22% in total biomass, while the addition of0.2 g of AMG to mycorrhizal pots resulted in a 3% decrease in biomass.Meanwhile adding 0.6 g of AMG to non-mycorrhizal pots gave a 45%increase, while adding 0.6 g of AMG to mycorrhizal pots gave only a 25%increase.

The results from the laboratory experiment lead to the followingobservations. In the ‘control’ situation, where no AMG was sown into thepots, the addition of mycorrhizae had a positive effect on the growth ofthe bentgrass. In pots where no mycorrhiza was added, then the additionof the AMG seed significantly increased biomass, as would be expected.However, if the mycorrhiza was present, addition of AMG at the level of157,000 seeds m⁻² had no effect on the total biomass and actuallyresulted in a small decrease. Indeed even at the highest level of sowing(471,000 seeds m⁻²), the mycorrhiza appeared to be able to mitigate theeffects of this seed addition. The seed additions in this experimentwere designed to be realistic compared with tiller densities in thefield. The presence of mycorrhizal fungi may serve to maintain AMG atlower levels than would occur if the fungi were absent. Furthermore,this mechanism can work at tiller densities comparable to thoseencountered on courses.

Field Study

A significant negative relation between AMG abundance and mycorrhizalcolonisation was again found ( F_(1.6)=8.65P<0.01, r²=0.351).Mycorrhizal colonisation was very low, compared with natural areas, butwas in the range (0-11%) The mean number of AMG tillers per core was75.9+/_(—)5.3.

Agrostis stolonifera in the course studied was virtually absent. Infact, 12 of the 18 greens appeared to be entirely AMG, and the mean AMGproportion in the sward was 96.1%. There was a significant differencebetween greens in the density of all crass tillers (c²=29.3, P<0.05) andalso in the proportion of AMG in the sward (c²=28.7, P<0.05). Therefore,the greens were not providing uniform surfaces, due to the variableamounts of AMG in them.

Greenhouse Trials Experiment 1 Effect of Vaminoc on Growth of Poa annua

8×1 liter trays, each measuring 20×15×5 cm were filled with 800 g of amixture of sandy soil (kettering loam from Rothamsted ExperimentalStation) and sand (1:1 w/w). 4 trays each were inoculated with 0.7 gVaminoc ( equivalent to 20 g/m⁻²), a three species mixture ofVA-mycorrhizal fungi (MicroBio Group Ltd., Whittlesford, Cambridge, UK).and 4 trays were left uninoculated. VAM inoculation was performed byraking Vaminoc lightly into the soil surface before seed sowing. Eachtray was sown with 1 g of Poa annua seeds, equivalent to the recommendedrate of 35 g/m⁻², and maintained in a glasshouse at 25/20 ° C. day/nighttemperature.

Grass shoots were cut 4 and 10 weeks after sowing and Shoot Fresh Weightdetermined. At the end of the growth period (10 weeks) the total weightswere calculated in each tray and root samples were stained and visuallyassessed for presence of VA-mycorrhizal colonization.

Results: FIG. 1 a and FIG. 1 b (photograph)

A significant reduction of growth of Poa annua in response to Vaminocinoculation as compared to uninoculated controls was observed. Thedetrimental effect of VA-mycorrhizal on Poa can be seen in FIG. 1b,taken after 10 weeks growth, before 2nd fresh weight determination.VA-mycorrhizal colonization was present in inoculated Poa but not inuninoculated control.

Experiment 2 Effect of Vaminoc on Poa annua under three P levels

It is well established that the growth response of plants to mycorrhizalinfection is influenced by the amount of phosphorus (P) supplied in thesoil. When P is readily available to the plant the positive growthresponse due to mycorrhizal infection is also reduced, and it has beenreported that high soil P levels reduce percentage infection inmycorrhizal plants.

Since VA-mycorrhiza have a negative effect on Poa annua, an experimentto study the relation between soil P levels and VAM response in Poa wasperformed by testing the effect of VAM inoculation under three P levels.

Materials and Methods were as indicated in Experiment 1.

Three soil P levels were chosen: no additional P(0), 152 g and 308 g ofTripleSuperphosphate (TSP) per tray (equivalent to 0, 100, and 240 kg/harespectively).

Therefore, there were 6 treatments (2 levels of mycorrhiza×3 TSPlevels), with 4 replicates of each, giving a total of 24 trays.

Shoot fresh weight was determined in each tray 4 and 9 weeks aftersowing. Total weights calculated after 9 weeks were analysed using aTwo-way ANOVA.

At the end of the experiment root samples were stained and checked forVAM fungal colonization. The results are shown in FIG. 2.

As expected, the overall weights for Poa annua trays not inoculated withVaminoc were higher than those for the inoculated trays(F_(1.18)=57.37;p<0.01). There was also a difference in the overallweights for the different levels of TSP (F_(2.18)=5.97; p=0.01).

Growth responses of Poa to the three TSP levels were different ininoculated as compared to uninoculated trays (F_(2.18)=8.87; p=0.002).In inoculated trays the growth retardant effect of Vaminoc was observedat all P levels tested.

At the end of the experiment. VAM colonization in roots of allinoculated Poa treatments were observed as compared to no colonizationin the uninoculated controls. Some mycorrhizal fungi are detrimentallyaffected by high soil phosphorus levels. When soil phosphorus isabundant, some fungal species grow very poorly and plant response isalso reduced. However, an important feature of the effect describedabove is that the antagonistic effect on Poa annua has been found atlow, medium and high soil P levels. The ‘high’ P level in thisexperiment corresponded to that found in typical putting green soil,where levels are invariably high. Thus, the antagonistic effect of thefungi on P. annua occurs in realistic situations, such as those found ingolf greens.

EXAMPLE 1

A practice putting green was used for the experiment. This was composedof a mixture of Agrostis spp. and Poa annua, with small amounts ofFestuca spp. The experiment took 6-7 months from late June onward.Twenty four plots, each 0.5 m×0.5 m were laid out on the green and eachplot was subjected to one of four treatments:

Addition of Agrostis seed, as an overseed, @4 g per plot

Addition of Vaminoc-T, @5 g per plot

Addition of seed and inoculum

Addition of neither (‘control’).

The plots were arranged randomly on the green, with six replicates ofeach treatment. Grass abundance was sampled non-destructively by thepoint quadrant method. Twenty 3 mm diameter metal pins were placedrandomly in each plot, so that the tip of each pin rested on the soilsurface. The total number of touches of each crass species on each pinwas counted, and summed over the 20 pins in each plot. Plots weresampled on five occasions over a six month period after the treatmentshad been applied. The average value was then calculated across the sixreplicates for each treatment on each date. The averages are displayedin the accompanying figures.

Results (see FIGS. 3, 4, and 5)

At the start of the experiment, the total amount of grass in eachtreatment was similar. Addition of seed had no effect on the totalamount of grass, or that of the two commonest species, Agrostis and Poa.However, by the end of the study, treatments which had the Vaminoc-Tmycorrhiza inoculant applied to them had significantly more total grasstouches (FIG. 3). The addition of the fungi therefore had stimulatoryeffect on sward growth with more grass blades being touched by the 20pins. However, the effect on the two dominant grass species was not thesame. The treatments in which Vaminoc-T was applied had more touches ofAgrostis at the end of the six-month period, as shown by the linesmarked with triangles in FIG. 4. Therefore, the fungi were much moreeffective in increasing abundance of this grass than was the addition ofseed.

Meanwhile, at the end of the experiment, plots which received Vaminoc-Thad fewer touches of Poa annua than did the plots in which no treatmentwas applied (FIG. 5). This was a complete reverse of the situation atthe start of the experiment, when ‘control’ plots had the lowest Poacount.

Therefore, application of Vaminoc-T to a working golf green resulted ina reduction in Poa annua abundance, an increase in Agrostis abundanceand an increase in overall grass abundance.

EXAMPLE 2 VAM Field Efficacy

For the purpose of this demonstration, two US PGA constructed greenswere selected from a large popular golf course in Cambridgeshire-UK. Thegreens were seeded approximately three years previous, and managed asper standard practice. Much of the green was composed of Agrostis spp.,with a low level of Festuca spp. Poa annua was evident across much ofthe two selected greens.

Each 500 m² green was split equally into two plots. The greens werehollow-tined prior to treatment. Vaminoc-G was applied at 20g m² to onehalf of each green, whilst the other half remained untreated. Each greenwas lightly irrigated within 8 hours of Vaminoc-G application.

Each green was visually assessed for P. annua incidence prior toVaminoc-G application, and again approximately 2 months afterapplication.

Greens were managed as standard practice during the course of the trial.

Results

The relationship between Vaminoc-G application and incidence of Poaannua for both greens are recorded on the accompanying FIGS. 6 and 7. Itis evident that although the incidence of P. annua generally declines inthe greens over the assessment period as expected, the total percentagereduction in the Vaminoc-G treatment areas is far higher than thatrecorded in the non-treated areas (96% and 84% cf. 59% and 34% for green1 and 18 respectively).

What is claimed is:
 1. A method of retarding growth of Poa annua ingrass containing at least a small amount of Poa annua, the methodcomprising applying VA mycorrhiza to the grass.
 2. The method of claim 1using one or more strains of VA mycorrhiza selected from genera Glomus,Acaulospora, Entrophosphora, Gigaspora, Scutellospora and Sclerocytis.3. The method of claim 1 using one or more strains of VA mycorrhizaselected from the group consisting of Glomus fasciculatum, Glomuscaledonium, Glomus mosseae, Glomus versiforme, Glomus intraradices andGlomus etunicatum.
 4. The method of claim 1 wherein the grass isturfgrass.
 5. The method of claim 4 wherein the turfgrass is one of a. agolfing green, or b. an amenity turf.
 6. The method of claim 4 whereinthe major portion of the turfgrass is formed of at least one of AgrostisSpp. and/or Festuca Spp.
 7. A method of controlling or reducing thegrowth of Poa annua in turfgrass which comprises applying to theturfgrass infective propagules of one or more strains of VA mycorrhizawhich are growth-controlling for Poa annua.
 8. The method of claim 7wherein the infective propagules are selected from fungal spores,mycelium, hyphae, and fragments of mycorrhiza infected roots.
 9. Themethod of claim 7 wherein the one or more strains of VA mycorrhiza areselected from genera Glomus, Acaulospora, Entrophosphora, Gigaspora,Scutellospora and Sclerocytis.
 10. The method of claim 7 wherein the oneor more strains of VA mycorrhiza are selected from the group consistingof Glomus fasciculatum, Glomus caledonium, Glomus mosseae, Glomusversiforme, Glomus intraradices and Glomus etunicatum.
 11. The method ofclaim 7 wherein the infective propagules are formulated with asubstantially chemically inert solid carrier.
 12. The method of claim 11wherein the solid carrier contains at least one of clay, silica gel,bleaching earths, pumice, bauxite, attapulgite, vermiculite, calcinedmontmorillinite, soil, peat, or sand.
 13. The method of claim 7 whereinthe infective propagules are applied to the turfgrass as a part of aformulated product wherein the propagules constitute the principalcomponent having biological activity on the growth of turfgrass.
 14. Themethod of claim 13 wherein the formulated product has an MPN of fromabout 200 to 1000 or more propagules per gram.
 15. The method of claim13 wherein the amount of formulated product applied to the turfgrass isfrom about 5 g/m2 to about 1 kg/m2.
 16. A method for the control of thegrowth of Poa annua in grass comprising applying to the grass acomposition which contains one or more strains of VA mycorrhiza and issubstantially free from any biological agent having direct or indirectPoa annua growth-promoting activity.
 17. The method of claim 16 whereinthe one or more strains of VA mycorrhiza are selected from generaGlomus, Acaulospora, Entrophosphora, Gigaspora, Scutellospora andSclerocytis.
 18. The method of claim 16 wherein the one or more strainsof VA mycorrhiza are selected from the group consisting of Glomusfasciculatum, Glomus caledonium, Glomus mosseae, Glomus versiforme,Glomus intraradices and Glomus etunicatum.
 19. The method of claim 16wherein the composition contains a solid carrier chosen from at leastone of clay, silica gel, bleaching earths, pumice, bauxite, attapulgite,vermiculite, calcined montmorillinite, soil, peat, or sand.